Y-Chromosome Determination
Over the years a number of methods have been developed for making a determination either directly or indirectly of the presence of the Y chromosome in a particular tissue sample.
Barr Body Test. The barr body test is an indirect method for determining that an XY karyology may be present in a tissue sample. Buckle smear and other tissue samples dyed with Giemsa reagent stain reveal the presence of any X chromosomes beyond one copy per cell. These chromosomes appear as barr bodies within the cell nucleus.
Unfortunately, the buckle smear method is not always accurate in predicting the presence of a Y chromosome. For instance, an XO genotype would give the same results as an XY genotype. Also, at various stages in the cell growth and division cycle or in certain tissue samples, barr bodies fail to become visible. Therefore, barr body testing is generally limited to non-critical screening assays.
Because of the limitations of barr body analysis, this method is not appropriate for use in invasively sampled fetal cells, such as chorionic villi or amniocentesis samples. The invasive sampling techniques used are expensive and not without risks. Therefore, multiple sampling is generally unacceptable, as are delays in re-diagnosis which can occur when test results are equivocal. Even a low level of false diagnosis is unacceptable in situations where prenatal gender would be the basis of pregnancy termination.
Karyology. With the advent of tissue culture techniques, direct karyology of cells became possible. However, there are several limitations to this technique. The sample tissue must be in an active growth phase during analysis to be useful in karyology. This is because the sample cells must be dividing for the chromosomes to be arrested in their condensed, visible metaphase stage.
In the case of mammalian tissue sample growth systems such as are required in conventional human prenatal diagnosis, karyology techniques have proven to be time consuming and expensive. Mammalian cell lines are highly fastidious in their culturing parameters and cell media requirements. Further, any contamination of mammalian tissue culture material can result in complete failure of growth of the cells.
Nucleic Acid Probes
The recent development of relatively Y specific DNA probes has great potential for many clinical, animal husbandry, forensic and paleontological applications, among other uses. Small or deteriorated tissue samples can be analyzed as long as a minimal amount of DNA can be obtained. If linked with the PCR or other amplification technologies, such probes are potentially useful in forensic determinations. In suspected rape cases, the presence of severely decayed sperm or its genetic remnants might be detected by such methods. Hair and fragmentary tissue samples could also be typed for gender. A very small sample size might be largely conserved by this method allowing a large amount of sample material for other analytical work.
Forensic Science Uses. PCR and DNA probes have been used in recent efforts to genetically identify highly decayed remains of "the disappeared" in Argentina and match children born in prison with remaining family members. Using a Y probe as an initial screening tool in such work or related work is useful in conserving limited samples.
Gender Determination Uses. Y probes are also potentially useful in determining the sex of embryos for transplant if only a single sex is desired, such as in various animal husbandry uses. When combined with artificial fertilization, such techniques could be used to increase the desired gender of offspring by subjecting sperm samples to column or other elusion techniques in order to enrich the sample for Y or X bearing sperm. For instance, Y bearing sperm can be bound to a labeled probe, and then sorted for gender. The Y or X bearing sperm can be collected, and utilized for insemination when male or female offspring are desired.
Presently Available Probes. The applicability of the presently available Y DNA probes are limited because the probes are only relatively Y chromosome specific. These probes have a comparatively high level of nonspecific binding to other chromosomes. This lack of specificity is believed to be due in part to the large size and complexity of the binding sequence employed in such probes. This nonspecific binding often varies from sample to sample, and so has an unpredictable impact on the sensitivity of the test in any particular situation.
In testing situations where the amount of Y chromosomal material is high compared to that of the other genomic DNA, the nonspecific binding inherent in prior art DNA Y probe systems is generally not critical to the success of the analysis. However, in other testing environments, the nonspecific binding of the probes results in the limitation of the applicability of the assay, or even forecloses its workability altogether. As an example, in the case of seeking Y chromosome bearing fetal cells in maternal blood, conventional DNA probes are ineffective. This is because conventional Y probes non-specifically bind to a high proportion of maternal genomic DNA. The signal from this non-specific binding obscures the signal from the binding to the very minute proportion of fetal DNA in the maternal blood.
There is a recognized need for probes with the capacity to bind exclusively to the Y chromosome. Presently available Y chromosome nucleic acid probe systems could be substantially improved by substituting such specific probes for the conventional probes now being employed. A technique by which a variety of such highly specific Y probes could be produced would allow a further improvement of these analytical systems. With such a technique, the most appropriate probe for any particular application could be developed.
Y chromosome specific nucleic acid probes made up of multiple copies of Y specific sequences would also have a number of advantages over the probes of the prior art. Probes of any particularly advantageous size could be manufactured. Additionally, substantial degradation of the probe's hybridizing nucleic acid strand could be suffered without loosing a large portion of the probe's annealing capacity.
Currently, there are no RNA probes for Y chromosome specific RNA products. However, such probes would be very useful for investigation of Y chromosome specific gene expression.
Polymerase Chain Reaction. In the past, DNA probes had limited applicability when the sample size of the target DNA was below detectable levels for a particular probe system. Detection of low sample sizes can now be accomplished by amplifying the desired sequence using polymerase chain reaction (PCR) techniques developed by various researchers (Mary, "Multiplying Genes by Leaps and Bounds," Science, Vol. 340, pp. 1408-1410, 1988). Such techniques have been used successfully in other areas of prenatal diagnosis such as in sickle cell anemia (Saiki et al., "Enzymatic Amplification of Beta-globin Genomic Sequences and Restriction Site Analysis for Diagnose of Sickle Cell Anemia," Science, Vol. 230, pp. 1350-1354, 1988). In the prenatal diagnosis materials successfully employed at present, the determination is one of relative levels of a DNA sequence in fetal cells in the presence of a small number of maternal cells. Materials are obtained through amniocentesis or chorionic villus sampling.
Prenatal Diagnosis of Fetal Gender
There are many clinical and social reasons for testing for fetal gender. There are a variety of clinical reasons for conducting such tests. It is useful to determine the sex of the fetus when there is a family history of sex-linked genetic diseases such as hemophilia. Gender determination would be helpful in such cases when planning neonatal and prenatal care and when making decisions concerning possible pregnancy termination. Such a diagnostic tool would be useful when there is a family history of such sex-linked genetic diseases as Lesch-Nyhan syndrome, Fabry disease, Hunter syndrome, Duchenne muscular dystrophy, nephrogenic diabetes insipidus, and glucose-6-phosphate dehydrogenase deficiency, among others.
There are other clinical applications of prenatal sex determination. Fetal gender determination is helpful to neonatologists and obstetricians in making judgments as to what treatment regimens are appropriate in particular cases. Gender determination can be a valuable clinical tool because of the greater maturity and survivability of female fetuses as compared to male fetuses of the same size and gestational age. For instance, fetal gender determination is useful when timing labor induction for fetal distress or for other reasons. It is helpful to have determined fetal gender when deciding to allow pre-term labor to proceed. Fetal gender identification can figure into an evaluation for potential lung maturation problems or other post-term risks.
Prenatal sex determination is important information for families with a strong gender preference. This is particularly true in countries where opportunities for women are very limited, and infanticide of female newborns is both historically and contemporarily practiced. Such practices could be dramatically diminished if early fetal sex determination were available with the option for a first trimester abortion.
Presently available techniques for fetal sex determination have a number of drawbacks which severely limit their use. These techniques are often suitable only for the later stages of pregnancy. They also require direct sampling of fetal tissues through cell collection from the amniotic fluid or the chorionic villus. Some success in fetal gender determination has been achieved by visualization of the fetus with ultrasound. Efforts have been made to test increased testosterone levels in the maternal blood as an indicator of fetal gender, but the results of this area of research have been inconclusive.
Amniocentesis. The advent and standardization of the amniocentesis procedure has resulted in the development of the now well established tissue culture technique for the analyses of fetal chromosomes. In this method, fetal lung and epidermal cells which have sluffed off into the amniotic fluid are sampled by the use of a large gauge hollow needle and a syringe. The cells are grown employing mammalian tissues culture techniques. Once sufficient growth is accomplished, the dividing cells are trapped in metaphase by the use of the spindle fiber poison colcemid. The resulting metaphase cells cultures are subsequently fixed, mounted, stained and photographed. Karyology, that is identification and grouping of the chromosomes, is then required to determine the sex of the fetus.
The amniocentesis method of gender determination has several drawbacks. Because there is no known treatment for most of the genetic diseases being tested for, the pregnancies in which affected individuals are predicted are often terminated. However, the test can only be accomplished in the second trimester of pregnancy due to lack of sufficiently developed fetal cells in the amniotic fluid in the earlier stages of fetal development. Pregnancy termination is considerably more complex at this stage, requiring more intervention, and with a greater risk of morbidity and mortality to the mother. Additionally, there are considerably more emotional problems to the family when pregnancies are terminated so late in the gestational period.
Although amniotic fluid sampling has been widely practiced with minimal complications, some degree of infection and other sequela have been associated with this sampling method. The expense of the diagnostic procedure is considerable, in part because eucaryotic cell culture techniques must be used for sample processing. The techniques require special laboratory facilities. Also, the cell culturing procedure requires considerable amounts of the time of skilled laboratory personnel to be reliably successful.
If the cultures of fetal cells become contaminated or fail to grow in some other way, the amniocentesis sampling procedure must be repeated. This can result in time delays which can put the pregnancy beyond the allowable period for a therapeutic abortion.
Because of the expense and complexity of this testing method, amniocentesis with cell culture and karyology is not available as a general screening tool. Candidates for the procedure often must journey to large metropolitan areas to have the test done. Screening for gender preference reasons is routinely denied to families because of the limited availability of the procedure, and the number of medically necessary cases requiring this limited resources. The entire process requires several weeks to obtain the needed information and generally costs over $500. Thus, this method is not useful in many of the other clinical situations enumerated above. Karyology and thus much of the expense and time cost of this procedure could be eliminated by the use of a reliable Y probe with a strong signal.
Chorionic Villi Sampling. A new prenatal diagnostic technique using the sampling of chorionic villus has been recently introduced. Although there is an increased risk of miscarriage from the procedure as compared to standard amniocentesis sampling techniques, chorionic villus sampling allows testing in a somewhat earlier stage of the pregnancy.
Unfortunately, the expense and commitment of laboratory staff and facilities to sample processing required by chorionic villus analysis is similar to the well established amniocentesis method. As with the amniocentesis sampling method, facilities equipped to process the samples are virtually unavailable to third world countries. The transport of the samples to appropriate testing centers is prohibitively expensive. Sadly, the countries which cannot provide such services are also those suffering from some of the highest rates of infanticide. As with amnio center is, such of the expense and waiting period required in this method could be eliminated by the use of a reliable Y probe with a strong signal.
Ultrasound Diagnosis. With the advent of ultrasound techniques in obstetrical practice, the gender determination of fetuses in late pregnancy has become a standard event in many pregnancies. However, relying on such visualizations as the basis for pregnancy termination is not standard medical practice. Even in the newborn infant, sex determination by physical observation can be highly variable due to a number of different factors such as localized edema. Additionally, as is the case with amniocentesis and chorionic villus sampling, the determination is made very late in the pregnancy with all of the incumbent disadvantages and even outright legal bars to pregnancy termination.
In certain academic radiology departments, there have been some claims that fetal gender can be determined by ultrasound methods as early as 11 weeks of gestation. However, it is generally accepted that such determinations cannot be reliably made before 16 gestational weeks even in an academic setting with state of the art equipment and a highly skilled radiologist making the determination. Even after 16 weeks gestational age, false determinations of female gender are possible.
DNA Probes. Some advances in the detection of Y chromosomal DNA have been made in the last few years by the use of DNA probes which display homology to various regions of the Y chromosome. However, none have been applied to the prenatal determination of gender with the possible exception of direct assays of fetal tissue as described above.
Prior art techniques for producing Y "specific" DNA probes are applicable only to testing requirements that allow some homology and binding of the probe to certain autosomal and X sequences. Thus, these prior art probe sequences have proven to be Y preferring rather than truly Y specific. RNA probes specific for Y chromosomal products have not been developed at all.
The conventional Y DNA probes preference binding is appropriate and workable where there is no significant degradation of the test material, and large amounts of Y containing DNA material are present. For instance, such tests would be applicable to making determinations of testicular feminization or other sex chromosome anomalies in children and adults. However, as a practical matter, such a role is filled by the inexpensive barr body test using a standard Giemsa reagent stain to identify a second X chromosome in fixed cells.
Where a sample of fetal tissue is taken directly from the amniotic fluid in the form of discarded epithelial cells, or where a portion of the chorionic villus is sampled, these new probes may potentially provide a method of determining fetal gender without resorting to expensive, time consuming cell culture or karyology technique, or as a confirmation to a borderline barr body test.